Fine needle aspirates from breast lesions In previous studies, we clearly demonstrated that detection of chromosomal aneuploidy and gene copy number changes could serve as a sensitive and specific test for the diagnosis of breast cancer in minimally invasive fine needle aspirates. The clinical value of this diagnostic test would be further enhanced if premalignant lesions could be stratified into those requiring treatment and those that would not. We are working together with the Breast Cancer Clinic at the Naval Medical Center to recruit patients and accrue samples of these preinvasive lesions for FISH analysis with the probe sets we previously developed. The results will then be prospectively validated on fine needle aspiration samples provided by the Breast Care Center at Navy. Cervical cytology (Pap-smears) CGH analyses of human tumors have revealed that chromosomal aberrations result in genomic imbalances specific for different tumor from diverse tissue types. Furthermore, these changes define discrete steps in the progression of epithelial tumors. More than 90% of cervical carcinomas carry extra copies of chromosome 3, which results in the genomic amplification of the human telomerase gene TERC. It is therefore logical to apply the visualization of these recurring and specific chromosomal aberrations to complement and enhance the cytomorphological diagnosis of human cancers and their precursor lesions. This can be achieved using interphase cytogenetics with fluorescently tagged DNA probes that recognize specific chromosomal target regions directly in interphase cells. The application of this genetic test to routinely collected cytological specimens proved that high-grade intraepithelial lesions (HSIL, which are comprised of CIN2 and CIN3) could be discerned from normal samples, ASCUS and low-grade intraepithelial lesions (LSIL, or CIN1) with a sensitivity and specificity exceeding 90%, independent of the cytomorphological assessment. In order to explore whether gain of 3q and genomic amplification of hTERC increases the propensity of progression from LSIL to HSIL and invasive carcinoma, we have applied the probe set to a series of previously stained PAP-smears. The samples included (i) CIN1 and CIN2 lesions that progressed to CIN3, (ii) CIN1 and CIN2 lesions that regressed spontaneously, and (iii) normal PAP-smears from women who subsequently developed CIN3 or cervical cancer. Our data suggest that genomic amplification of hTERC is required for the transition from CIN1 and CIN2 to CIN3 and that it predicts progression. Of note, hTERC amplification was found in 30% of cytologically normal PAP-smears from women who were diagnosed with CIN3 or invasive cervical carcinomas after a short latency. We conclude that the detection of genomic amplification of hTERC in routinely collected PAP-smears can assist in identifying low-grade lesions with a high progression risk and in reduction of false negative individual cytological screenings. In an independent study we were curious as to whether the amplification of the human telomerase gene could also be used to diagnose another type of cervical cancer, i.e., cervical adenocarcinomas. Interphase nuclei prepared from archival material of 12 primary cervical adenocarcinomas were hybridized with a triple colour probe-set specific for centromeres of chromosomes 3 and 7 and the TERC gene. The findings were correlated with HPV infection. We observed high proportions of nuclei with increased copy numbers for TERC in both HPV positive and HPV negative tumors. The absolute number of TERC signals was increased in HPV positive (mean 3.7) and HPV negative (mean 5.0) tumors. Amplification of the human telomerase gene TERC is a consistent aberration in cervical adenocarcinomas and is independent of the presence of HPV genomes. Therefore, application of our probe set may provide an objective genetic test for the assessment of glandular cells in Pap smears and hence for the diagnosis of cervical adenocarcinomas. Based on the results discussed in 3.1.1, we designed a large-scale validation study in which the presence of TERC amplification in Pap smears was correlated to the histological diagnosis after colposcopy and biopsy. In this study, we used a four-color FISH assay by Abbott/Vysis based on our previous results that included a biotinylated DNA probe for the detection of HPV (provided to us through a MTA with that company). In collaboration with the Karolinska Hospital in Stockholm, Sweden, we collected 78 liquid based cytology (LBC) samples from women referred to the Karolinska Hospital because of a suspicious Pap smear. At the time of LBC sampling, all patients were examined by colposcopy, at which time a biopsy was taken. For most patients a subsequent histological diagnosis after loop electrosurgical excision procedure (LEEP) and a follow-up Pap smear were available. The LBC samples were also analyzed by several HPV tests (line blot, Quantovir HPV test, HPV mRNA detection using the NorChip assay), and for the expression levels of p16 using immunocytochemistry. The histological diagnosis revealed 14 benign cases, 17 samples rated as cervical intraepithelial neoplasia (CIN) grade 1, 22 CIN2, 23 CIN3, and two carcinomas. As expected, the number of TERC positive cases increased with the severity of the dysplasia. We then compared, using Receiver Operating Characteristic (ROC) curves, which of the tests rendered the highest sensitivity and specificity for the discernment of benign lesions and CIN1 from CIN2, CIN3, and cancer. We showed that detection of genomic amplification of TERC clearly outperforms any other test, including detection of HPV, as a diagnostic test for the presence of high-grade cervical dysplasia (CIN2 and CIN3) and cancer in routinely collected LBC samples. The correlation with the histological classification, the standard for a diagnosis, was excellent. In collaboration with Pamela Paris (UCSF) we will also study prostate tissue sections for copy number changes of a high-risk diagnostic probe cocktail which was defined after aCGH analyses of prostate carcinomas associated with a high progression risk.